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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 238-243
VH Gene Analysis of Clonally Related IgM and IgG From
Human Lymphoplasmacytoid B-Cell Tumors With Chronic Lymphocytic
Leukemia Features and High Serum Monoclonal IgG
By
Surinder S. Sahota,
Richard Garand,
Regis Bataille,
Alastair J. Smith, and
Freda K. Stevenson
From the Molecular Immunology Group, Tenovus Laboratory, Southampton
University Hospitals, Southampton, UK; and the Centre Hospitalier
Universitaire de Nantes, Institut de Biologie, Nantes Cedex, France.
 |
ABSTRACT |
An unusual group of human B-cell tumors with cellular features of
chronic lymphocytic leukemia or lymphoplasmacytoid leukemia, together
with high levels of a monoclonal IgG serum protein, has been
investigated. Analysis of tumor-derived VH genes of
neoplastic B lymphocytes was used to determine the clonal relationship
between the IgM expressed or secreted by the tumor cells and the IgG
serum paraprotein. In all five cases, VH gene sequences
showed transcripts of IgM and IgG of common clonal origin. Sequences
were derived from VH3 (4 of 5) and VH1 (1 of 5)
families and were all highly somatically mutated with strong evidence
for antigen selection. There was no intraclonal variation detectable in
either IgM or IgG sequences. In 3 of 5 cases, in which monoclonal IgM
and IgG were found in serum, the VH genes combined to Cµ
or C showed identical mutational patterns. However, in 2 of 5 cases,
in which IgM was confined to cell expression with only monoclonal IgG
in serum, sequences of the VH transcripts of IgM and IgG
showed many shared mutations but also numerous differences. In these
cases, the level of mutation was similar in IgM and IgG and both
appeared to be antigen selected. In summary, the final neoplastic event
in this group of tumors has apparently occurred at the point of isotype
switch from IgM to IgG, leading to dual isotype synthesis. In the group
that secreted both isotypes, the mutation pattern was identical,
indicating either synthesis by a single cell, or silencing of
mutational activity before switching. In the group that did not secrete
IgM, cells of each isotype were distinct and reflected a divergent
mutational history.
| |
INTRODUCTION |
DURING NORMAL B-cell differentiation, a
functional VH-DH-JH transcript is
generated by a process of genetic recombination. A single
VH gene is chosen from the available VH
repertoire consisting of approximately 51 potentially functional genes
that can be grouped into structurally related families VH1
to VH7.1 Choice of D-segment gene and
imprecision at the joints gives rise to heterogeneity in amino acid
sequence at the third complementarity-determining region (CDR3).
Therefore, the CDR3 sequence can be considered as a clonal signature
for a B cell and, following neoplastic transformation, can act as a
useful tumor marker.2
After a similar process of recombination in the light chain, a naive B
cell expresses IgM and can bind antigen. The cell may then enter the
lymphoid follicle and undergo a second process of sequence
diversification by somatic hypermutation.3 Although the
introduction of mutations has an element of randomness, the limited
antigen in the germinal center will stimulate only those B cells that
offer complementary sequences to antigen via the contact points in
CDR1, CDR2, and CDR3.3,4 Therefore, the imprint of antigen
selection will be a clustering of replaced amino acids in these
regions. The next process in a maturing antibody response is a switch
from IgM to another isotype, often IgG. The switching mechanism has
been shown to involve deletional recombination with excision of the
upstream constant regions.5 It appears that a considerable
degree of somatic mutation can occur before switching,6,7
leading to production of IgM memory cells.7 Further
mutations may accumulate after switching,6,7 but the
mutational mechanism appears silent in the fully differentiated plasma
cell.
Neoplastic transformation can occur at several points in B-cell
differentiation, from the pre-B cell to the plasma cell. Investigation
of VH genes used by B-cell tumors is now providing
information on the nature of the cell of origin and its clonal history.
For example, it appears that the usage of VH genes by
certain tumors is highly asymmetric, which may be important for
pathogenesis.8-10 Analysis of VH gene sequences
is also indicating whether the cell of origin has entered the germinal
center, known to be the site of somatic mutation.3
In the case of chronic lymphocytic leukemia (CLL), a proportion of
VH sequences are unmutated, consistent with a naive B
cell.11 However, there are subsets of CLL, particularly
those with abnormalities in chromosome 13q14, that have accumulated
significant levels of mutation, indicative of a different maturation
state.12 Follicular lymphoma and Burkitt's lymphoma have
both undergone somatic mutation as expected, and mutations continue to
accumulate in these tumors subsequent to the final transformation event
leading to intraclonal heterogeneity.13-15 In contrast,
splenic lymphoma with villous lymphocytes16 and
myeloma17 have mutated VH sequences, but no
intraclonal heterogeneity, suggesting that the final event in these
tumors is at a postfollicular stage.18
Although it is possible to induce isotype switching in CLL cells in
vitro,19,20 the ability of B-cell tumors to isotype switch
in vivo was difficult to document before the development of
VH gene probes. Coexistence of CLL and an isotype-switched
plasma cell tumor such as myeloma is very rare, and commonly involves
different light chain types, or different idiotypic antigens,
indicative of biclonality.21 In an extensive literature
survey of CLL and the more aggressive tumors that develop in
approximately 10% of patients, genetic relatedness was a common
finding only in cases of prolymphocytic transformation.22
Transformation to myeloma showed clonal relatedness in only 2 of 8
cases, indicating that this type of disease progression arises
relatively rarely via clonal evolution,22 a finding
supported by more recent cases.23 However, transcripts of
clonally related VH-Cµ have been found in patients with
myeloma, which point to coexistence of a minor population of less
mature B cells.24,25
We have had the opportunity to study an unusual subset of B-cell tumors
with abnormal cells expressing or secreting both monoclonal IgM and
IgG. Such cases are likely to be closer to lymphoplasmacytoid lymphoma
(LPL) than to true CLL, although some share features with both
entities. LPL is a distinct B-cell proliferative disorder with
particular morphological and histopathological characteristics, and is
frequently associated with a high secretion of monoclonal
IgM.26 However, cases of CLL may also exhibit high levels
of monoclonal Ig, usually IgM, and can show morphological or phenotypic
lymphoplasmacytoid differentiation.27 On the other hand,
cases of LPL may have blood and bone marrow involvement with cells
difficult to distinguish from CLL.26 Our cases appear
closer to LPL than CLL and have the additional rare feature of
synthesizing both IgM and IgG. Because they may, therefore, reflect the
point of isotype switch, we have used immunogenetic analysis to show
the clonal history of these tumors.
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MATERIALS AND METHODS |
Patient selection.
The focus of this investigation was on patients with B-cell tumors who
had evidence for synthesis of both IgM and IgG. From January 1989 to
May 1996, we investigated in the Department of Hematology in Nantes,
France, 611 cases of B-cell lymphoproliferative disorders (BLPD), with
circulating abnormal lymphoid cells >5 × 109/L, and
CD19 >30%. Among these, we observed 12 patients with a high serum
monoclonal IgG. Four of these patients had the same light chain type in
IgM and IgG and were selected for further study. An additional patient
(BAR) presented in Southampton with renal stones, and was found to have
IgM and IgG paraproteins of similar light chain type in serum. He had a
normal blood count and has remained well for 6 years since diagnosis.
Morphological and histological classification of BLPD.
All May-Grünwald-Giemsa stained blood and bone marrow smears were
reviewed and classified according to the French-American-British (FAB)
group proposals.28 Similarly, all biopsy specimens were
reviewed and classified according to the revised European-American
classification of lymphoid neoplasms (REAL).26
Immunophenotypic analysis.
Peripheral blood mononuclear cells were prepared by separation on a
Ficoll gradient and analyzed by indirect immunofluorescence, using a
panel of monoclonal antibodies (MoAbs), including B3 and B6 (CD22 and
CD23, respectively; Coulter, Miami, FL), leuM5 (CD11c) and B-B4
(CD138), a SYNDECAN-1 plasmacyte-specific MoAb29 (Diaclone
Research, Besancon, France), using the FACSCAN (Becton Dickinson, San
Jose, CA). Coexpression of CD5 and CD19 was assessed
by double labeling using fluorescein isothiocyanate (FITC)-labeled
IOT1a (CD5) and phycoerythrin (PE)-labeled IOB4 (CD19; Immunotech,
Marseille, France). Surface Ig (sIg) expression was studied with
FITC-labeled F(ab )2 from goat polyclonal antibodies
against human Igµ, Ig , Ig, Ig , and Ig chains. sIg
fluorescence intensity was considered as strong when the difference
between mean fluorescence of both anti-light chain types exceeded one
log.
Analysis of VH genes.
Total RNA (5 to 50 µg) was isolated using RNAzol (Cinna Biotecx Labs
Inc, Houston, TX) from mononuclear cell fractions prepared either from
heparinized peripheral blood and stored as cryopreserved cells in
dimethyl sulfoxide (0.9 to 1.5 × 107 cells; patients
PAI, BLO, SAM, and LAR), or from a heparinized bone marrow aspirate
(patient BAR). RNA (2 to 10 µg) was reverse transcribed to cDNA with
Cµ and C isotype-specific primers as described.30 A
sample of cDNA was then amplified by the polymerase chain reaction
(PCR) using a mixture of 5-oligonucleotide primers that cover
the VH1 to VH7 family potentially functional
repertoire, together with nested downstream 3-primers specific
for the CH isotype. Amplification conditions and methods of
cloning and sequencing of products have been described.30
| |
RESULTS |
Clinical and laboratory features of patients.
All patients had evidence of B-cell tumors that were synthesizing IgM
and IgG, but the detailed nature of the tumors was variable. Four of
five patients presented with a significant lymphocytosis with cell
morphology typical of CLL. However, a few coexisting lymphoplasmacytoid
cells were observed in two patients (BLO and SAM). One of five patients
(BAR) had no lymphocytosis but had tumor cells in the bone marrow with
morphology consistent with LPL. Histologic data were available in three
of five cases (Table 1). According to the
REAL classification, two patients showed a histopathologic picture of
LPL, one in a bronchial biopsy specimen (BLO), the other in bone marrow
and in spleen (red pulp infiltrate; SAM). A third patient (LAR) had an
amyloid immunoglobulin light chain (AL)-type amyloid nephropathy on a
kidney biopsy specimen. All patients had high levels of monoclonal IgG
paraproteins in serum, and in one case (PAI) two IgG paraproteins were
detected. Patients PAI, BLO, and BAR also had an additional IgM
paraprotein of the same light chain type as the IgG
(Table 2). In four of five cases,
circulating tumor cells were present, and phenotypic analysis
(Table 3) showed increased numbers of
CD19+ lymphocytes. Only one case (PAI) showed an
immunophenotypical profile characteristic of CLL (CD5+
CD22+ CD23+), and these cells had an unusual
strong sIg (++) expression (Table 3). Patients BLO, SAM, and LAR
exhibited a CD5 CD22+
CD23+/, sIg++ surface phenotype more
typical of LPL.26,31 The four cases with tumor cells in the
blood were all negative for CD10, CD11c, and B-B4.
Circulating lymphoid cells expressed both sIgM and sIgG with similar
light chain type in three patients (PAI, SAM, and LAR), but only IgM in
patient BLO. In the case of BAR, only a bone marrow aspirate was
available, and this had the cytological features of LPL. The tumor
cells expressed CD19, and analysis of cytoplasmic Ig showed an
IgMG+ population. However, in all cases, conclusions
concerning dual expression of IgM and IgG were limited by the presence
of high serum IgG paraprotein, which may be bound to Fc
receptors.32
VH genes of tumor-derived VH-Cµ and
VH-C transcripts.
Identification of expanded tumor-derived VH sequences from
repeated identical or closely similar CDR3 "clonal signature"
sequences, obtained by PCR/cloning, has been validated
previously.33 In all cases, amplification of VH
in combination with C yielded repeated sequences with clear CDR3
"clonal signatures" (Table 4).
Tumor-derived deduced amino acid sequences are shown in
Fig 1. Nucleotide sequences have been
deposited in the European Molecular Biology Laboratory (EMBL) database
(accession numbers: Z95481-95487). Other VH sequences
obtained were individually different and were likely to be from normal
B cells. For all patients, the same VH sequence was also
found to predominate in sequences combined to Cµ (Fig 1), indicating
derivation of both IgG and IgM from the same original B cell. Most of
the tumors used VH genes from the VH3 family (4
of 5), with one out of five from VH1. The CDR3 sequences
were short (6-14 amino acids), particularly when compared with typical
CLL,11 and a range of JH genes was used, some
of which were somatically mutated (Fig 1). There was no evidence for
intraclonal variation in any of the tumor-derived sequences. All the
IgG products were derived from the IgG1 or IgG2 subclass (data not
shown); further assignment could not be made from the limited available
length of constant region nucleotides.
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| Fig 1.
Deduced amino acid sequences of VH-C and
VH-Cµ transcripts from patients' tumor cells
synthesizing both IgG and IgM isotypes. (A) Cases with both IgM and IgG
serum parapoteins. (B) Cases with IgG serum paraproteins only.
Comparisons are made with the closest germ line VH genes.
Upper case, replacement mutations; lower case, silent mutations.
Replacement mutations in JH are underlined.
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|
In the case of PAI, although two IgG paraproteins were observed in
serum, only a single VH-C transcript was identified,
suggesting that either the two IgG serum paraproteins were clonally
identical, or that the second paraprotein was being produced from a
separate cell population. In contrast, two different repeated
VH-Cµ transcripts were found for PAI, one of which was
identical to the IgG-derived sequence (Table 4). The VH of
the second IgM-derived transcript used the V4-59 gene
segment but contained a stop codon at position 47 rendering it
aberrant. This aberrant sequence was likely derived from the allelic
chromosome of the tumor cells, and had apparently not undergone isotype
switching.
Analysis of somatic mutations.
To assess the degree of somatic mutation, the VH sequences
were compared with the closest germ line sequences in the databases.
Although this approach does not allow for sequence polymorphism, it has
become clear recently that the majority of polymorphic changes in human
VH genes arise from deletions or insertions, and that
sequence variation is at a low level.1 The percent homology
of the VH sequences with the closest germ line genes was
quite low (Table 4) indicative of extensive somatic mutation. Deduced
amino acid sequences (Fig 1) show the positions of the mutations and
indicate amino acid replacements. Comparison of the pattern of
mutations in the sequences derived from IgG with those from IgM showed
complete identity for patients PAI, BLO, and BAR (Fig 1A). In contrast,
the pattern of mutation in IgG and IgM from patients SAM and LAR showed
some common mutations but many differences, particularly in the CDRs
(Fig 1B). However, the level of mutational activity was similar for
both IgG and IgM, and there was no evidence for accumulation of
mutations in switching from IgM to IgG. In fact, at several codons,
there were mutations in the IgM-derived sequences, which were not found
in the IgG sequences.
There was an apparent clustering of replaced amino acids in CDR1 and
CDR2 in all sequences, and this was assessed for statistical
significance, which points to antigen selection, using the method of
Chang and Casali.34 The results
(Table 5) show highly significant
clustering of replacement amino acids in the CDRs for IgG- and
IgM-derived sequences from all patients. Where mutations differed
between IgG and IgM, both had strong evidence for antigen selection.
There was also evidence for preservation of sequence in all the
functional framework regions (FWRs; Table 5). A different pattern was
observed in the nonfunctional IgM-derived V4-59 sequence
from patient PAI (Table 5). In this aberrant gene, there was no
clustering of somatic mutations in CDRs indicative of antigen
selection, nor was there evidence for conservation of sequence in the
FWR.
| |
DISCUSSION |
Assessment of the maturational status of B-cell tumors has, until
recently, been based largely on morphological and phenotypical
features. LPL is considered to arise from a mature B cell, often with
cytoplasmic Ig, and commonly associated with a monoclonal serum
paraprotein, usually of IgM type.26 Waldenstrom's
macroglobulinemia is the most common lymphoma in this category and has
been described as a distinct disorder of small lymphocytes showing
maturation to plasma cells.26 However, many B-cell tumors,
including CLL, also show some maturation to plasmacytoid cells. These
observations suggest that B-cell tumors are not completely
"frozen" at a point of differentiation.
Analysis of VH and VL genes is allowing further
probing of the differentiation status of B-cell tumors. Because the
V-gene germ line repertoire is largely mapped,1 it is
possible to investigate the degree and pattern of somatic mutations
that have occurred in the cell of origin. This process is activated in
the germinal center; therefore, accumulation of mutations indicates
that the cell has encountered this site.3 However,
mutational activity can continue after neoplastic transformation, as
seen clearly in follicular lymphoma in which clonally related cells
have distinct intraclonal sequence variation.13,14 The
distribution of mutations may also indicate a role for antigen
selection, and, because antigen may persist,35 it could
stimulate growth and differentiation of tumor cells.36 The
cases we describe were characterized by circulating lymphoid cells with
CLL morphology, but shared histopathologic and immunophenotypical
features of LPL; thus, they may correspond rather to
"lymphoplasmacytoid" leukemia than to true CLL. They have
accumulated somatic mutations as expected from a mature cell, which has
evidently passed through the germinal center. In fact, clustering of
replacement amino acids in CDR1 and CDR2 is even more obvious than in
the closely related splenic lymphoma with villous
lymphocytes16 or in follicular lymphoma,13,14
Burkitt's lymphoma,15 or
myeloma.17,18,30,36,37 This significant clustering provides
strong evidence for antigen selection up to and possibly beyond the
final neoplastic event.
Isotype switch events in normal B cells appear to occur at about the
same time as somatic mutation6 leading commonly, but not
invariably, to mutated sequences in IgG+
cells.38 A similar heterogeneity in mutational frequency is
seen in the rare cases of IgG+ or IgA+
CLL.39-41 Interestingly, in some cases of IgG+
CLL, transcripts of Cµ and C combined to the tumor
VH-DH-JH sequence have been
observed.42 This suggests either that minor populations of
cells with variant isotype switch products exist, or that a single
population can generate alternative transcripts by trans-splicing of
RNA.43 In one report, the fact that tumor cells expressed
dual sIgG and sIgA would support the latter mechanism.40
However, the argument for separate populations is supported by
detection of a few mutational differences in transcripts in some
cases42 and by the demonstration of deletional bi-allelic
recombination in three cases of isotype-switched CLL.40
A further surprising finding is that blood lymphocytes from cases of
conventional IgM+ CLL contain transcripts of tumor VDJ
combined to a variety of alternative isotypes and that cells can be
induced to synthesize the switched Ig.20,44,45 Thus,
although isotype switching occurs rarely in CLL, transcripts are
present. It is not known at present if these are produced by the bulk
population or from minor populations that have undergone deletional
switch recombination, although again there is some evidence for the
latter.45
The cases we describe appeared to be arrested at a more mature point in
differentiation. They all had high levels of monoclonal serum IgG, and,
in three of five cases, an additional monoclonal IgM. Analysis of the
VH genes of these three cases showed transcripts of
identical VDJ sequences combined to both Cµ and C. For PAI and
BAR, there was no phenotypical evidence for two populations, but this
cannot be ruled out. For BLO, only IgM expression was found, suggestive
of a separate undetected minor population of isotype-switched cells.
The only unequivocal way of confirming isotype heterogeneity among cell
populations with identical VH sequences would be to
separate out individual tumor cells and perform PCR at the single-cell
level, and this is in progress. In the other two cases, transcripts of
VDJ-Cµ and VDJ-C were also obtained with CDR3 similarity
indicating clear common clonal origin, but there were numerous distinct
mutations. For these cases, the IgG+ and IgM+
populations must be separate. This is in spite of the finding that the
cells appeared to coexpress IgG and IgM, and confirms the dangers of
phenotypic analysis of sIg32 particularly in the presence
of high levels of serum IgG. All sequences had strong evidence for
clustered replacement mutations characteristic of antigen selection, a
feature lacking in the aberrant allelic VH gene identified
in one of the patients. Therefore, it appears that the transformation
event occurred at the level of an IgM+, somatically
mutated, antigen-selected B cell, with characteristics of a memory B
cell. The transformed cell proliferated giving rise to a neoplastic
clone, which, in two cases, secreted IgM. This cell might be analogous
to tumor cells of Waldenstrom's macroglobulinemia in which V-genes
have been found to be somatically mutated.46 However, in
our cases, the cell was also able to undergo isotype switching to IgG,
and a second tumor population was able to proliferate and differentiate
to high level IgG secretion. In the IgM-secreting tumors, if the IgM
and IgG are produced from different cells, the mutation mechanism must
have been silenced before switching. In tumors not secreting IgM,
mutation continued, possibly in both isotypes. This continuing
mutational activity led to mutational divergence, with accumulation of
some mutations in the IgM-derived sequences that were not found in the
IgG-derived sequences. Curiously, no intraclonal variation was
detectable, indicating that the neoplastic cells represent progeny of
only single IgG and IgM variants. It also suggests that the current
IgM+ population has lost the ability to switch. These
tumors appear to have undergone neoplastic transformation at the
pivotal point of isotype switch from IgM to IgG. They may in fact
represent the bifurcation leading to memory cells or plasma
cells,47 and the IgM+ cells could relate to the
precursor cell identified in some cases of myeloma.24,25
Therefore, immunogenetics has supported morphology in showing that
B-cell tumors can respond to differentiation signals in the same way as
normal B cells and has shown that the outcome can be two clonally
related tumor populations.
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FOOTNOTES |
Submitted June 5, 1997;
accepted August 28, 1997.
Supported by the Leukaemia Research Fund, UK, and the European Myeloma
Research Network (Biomed BMH1-CT93-1407).
Address reprint requests to Freda K. Stevenson, PhD, Molecular
Immunology Group, Tenovus Laboratory, Southampton University Hospitals,
Tremona Road, Southampton, SO16 6YD, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
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Abstract
Introduction
Abstract